A virus is a group of submicroscopic infective agents with double or single stranded DNA or RNA as core genetic material surrounded by a protein (and lipid in some case) shell called `capsid` or `coat`. It has no semipermeable membrane and it can multiply only in living cells using host cellular components. The short segment of the virus genetic material (FLt promoter) used in this invention can not infect plants or other organisms to cause disease. It is useful with selected foreign genes to obtain expression of these genes in other plants to confer useful properties to those transgenic plants.
Promoters from caulimoviruses
The following is a description of caulimoviruses also called plant pararetroviruses. Caulimoviruses derived their name from cauliflower mosaic virus (CaMV), the type member of the group (for reviews see Shepherd, 1989; Covey and Hull, 1992). More than a dozen types of caulimoviruses have been described to date. All have small circular DNA molecules as their genetic material. The genomes of CaMV (Gardner, et al., 1981) and four other members of this group, namely carnation etched ring virus (CERV), (Hull, et al., 1986), figwort mosaic virus (FMV), (Richins, et al., 1987) soybean chlorotic mottle virus (SoCMV), (Hasegawa, 1989), and peanut chlorotic streak virus (PClSV) (Richins, 1993; Richins, et al., 1995) have been fully sequenced. CaMV is a circular double stranded DNA virus with a genome size of approximately 8 kb. It is organized into seven open reading frames (genes) and two intergenic regions.
In the case of CaMV, the polypeptides corresponding to the six genes (I to VI) have been detected in infected cells and their functions have been identified. The cell-to-cell movement function (Thomas, et al., 1993; Ducasse et al., 1995), aphid-transmission factor (Daubert et al., 1983; Woolson, et al., 1983), minor capsid protein (Giband, et al., 1986), major capsid protein (Daubert, et al., 1982), reverse transcriptase (Takatsui, et al., 1992), and inclusion body protein (Odell and Howell, 1980) are associated with ORFs I to VI respectively. The gene VII protein was not detected in vivo (Wurch, et al., 1991). Its function is not clearly established. However a sequence located with this ORF of FMV is involved in translation of viral genes (Gowda, et al., 1991).
The viral genome is replicated through reverse transcription of the terminally redundant full length transcript (Bonneville and Hohn, 1993) by a virus encoded reverse transcriptase. Two major viral transcripts, known as 35S RNA and 19S RNA are synthesized exclusively from the minus strand DNA by the host RNA polymerase II (Odell, et al., 1981; Howell and Hull, 1978). The large intergenic region (L-IR) which resides between gene VI and VII, contains the promoter (35S) for the full length transcript which spans the entire viral genome (Dixon and Hohn, 1984; Scholthof, et al., 1992). The 35S RNA serve as template for minus strand DNA synthesis by viral gene V encoded reverse transcriptase (Gordon, et al., 1988). The small intergenic region (S-IR) residing between gene V and gene VI contains a promoter (19S) which transcribes gene VI only (Odell and Howell, 1980). The PClSV is apparently lacking the S-IR sequence, however both FMV (Scholthof, et al., 1992) and PClSV (Richins, 1993) have also been shown to have similar transcripts to the 19S and 35S RNA found in CAMV infected plant cells.
The CaMV 35S promoter, which spans about 941 base pair (bp) upstream from the transcription start site, has been shown to be active in various monocot and dicot cells. The cis-regulatory elements that are involved in directing transcription initiation reside within this region. The CaMV 35S promoter has a modular construction that includes an enhancer (Lam, 1994, and references there in) similar to those of other promoters like that of SV40 in mammalian systems (Ondek, et al., 1987; Schirm, et al., 1987; Fromental, et al., 1988). The 5' deletion analysis of CaMV35S promoter, studied in transformed tobacco calli or a protoplasts transient assay system, indicates that a promoter fragment of 343 bp upstream from the transcription start site is sufficient for high promoter activity (Odell, et al., 1985, Ow, et al., 1987).
The high CaMV35S promoter activity is the result of synergistic and combinatorial effects of enhancer elements residing in the -343 to -46 region upstream of the TATA element promoter (-46 to +8) (Fang, et al., 1989, Benfey, et al., 1989, Benfey and Chua, 1990, Benfey, et al., 1990a and Benfey et al., 1990b).
U.S. Pat. No. 5,378,619 to Rogers discloses a full length transcript promoter from the figwort mosaic virus. U.S. Pat. No. 4,940,835 to Shaw discloses the cauliflower mosaic virus 35S promoter. The latter patent claims chimeric plant genes containing the cauliflower mosaic virus promoter sequence. The patent does not disclose a double full length transcript promoter from the figwort mosaic virus in a transgenic plant and its expression advantages.
Several protein binding sequence motifs have been identified in the enhancer region of the 35S promoter (Lam, et al., 1989; Lam and Chua, 1989; Prat, et al., 1989; Bouchez, et al., 1989, Yanagisawa and Izui, 1992). Identical or similar sequence motifs are also present in promoters of other caulimoviruses (Bouchez, et al., 1989; Sanger, et al., 1990; Cooke and Penon, 1990; Richins, et al., 1993). Two nuclear binding protein factors, known as Activating Sequence Factor-1 and -2 (ASF-1 and ASF-2) from tobacco have been well characterized. ASF-1 binds to the activating sequence as-1 (-82 to -62) region of 35S promoter. Two TGACG motifs within this site are essential for DNA-protein interaction (Lam, et al., 1989). The as-1 motif is also found in full length transcript promoters from other caulimovirus including FMV (Sanger, et al., 1990, and present studies), PClSV (Richins, 1993) and MMV (Shepherd group, unpublished observation).
Single or multiple copies of enhancer sequences from the CaMV 35S promoter can increase homo- and heterologous promoter activity in an orientation-independent manner (Kay, et al., 1987; Ow, et al., 1987: Odell, et al., 1988; Fang, et al., 1989; Driesen, et al., 1993; Omirulleh, et al., 1993). The enhancement of promoter activity was proportional to the copy number of the enhancer sequence (Kay, et al., 1987; Ow, et al., 1987; Omirulleh, et al., 1993). Similar observation was made when single or multiple copies of the enhancer sequence was inserted upstream of the TATA element of the CaMV19S promoter (Ow, et al., 1987; Driesen, et al., 1993), rbcS-3A promoter (Fang, et al., 1989) and the nos promoter (Odel, et al., 1988).
U.S. Pat. No. 5,463,175 to Barry et al. discloses the figwort mosaic virus promoter. U.S. Patent No. 5,503,999 to Jilka et al. discloses the cauliflower mosaic virus 35S promoter and the figwort mosaic virus 35S promoter. U.S. Pat. No. 5,145,783 to Kishore et al. discloses the cauliflower mosaic virus 35S promoter. Figwort mosaic virus promoter is also disclosed.
U.S. Pat. No. 5,242,412 to Brown et al. discloses the figwort mosaic virus 35S promoter. U.S. Pat. No. 5,510,253 to Mitsky et al. discloses the figwort mosaic virus promoter. U.S. Pat. No. 5,512,466 to Klee et al. discloses the cauliflower mosaic virus 35S promoter and the figwort mosaic virus promoter.
U.S. Pat. No. 5,304,730 to Lawson et al. discloses the figwort mosaic virus 35S promoter. PCT Publication WO 94/24848 discloses a transgenic plant in which a chimeric gene comprising a wound inducible promoter which shows enhanced resistance to insect infection. Examples of vectors at least with a pKYLX4, pKYLX5 and pKYLX71 vectors.
U.S. Pat. No. 5,106,739 to Comai et al. discloses a caulimovirus 35S enhanced mannopine synthase promoter and method for using the promoter. The patent also discloses the use of a double CaMV 35S promoter in a construct used to create transgenic plants.
Proceedings of the National Academy of Sciences, Volume 90, page 6110-6114, July 1993, entitled "Plants that express a potyvirus proteinase gene are resistant to virus infection". This publication discloses pKYLX71:35S vector.
The Journal of Cellular Biochemistry, Supplement 16F, Apr. 3-16, 1992, discloses a binary vector PKYLX71-GUS. In Vitro Cellular & Development Biology, March 19, 1992, Volume 28, No. 3, Abstract P-1119 discloses PKYLX71-GUS vector. Molecular & General Genetics, Volume 220, page 389-392, Spring 1990, discloses expression of the caulimovirus 35S-GUS gene in transgenic rice plants.
Chemical Abstracts, Volume 119, Abstract No. 197251n discloses transgenic plants with increased solids content. The plants are made with a construct including a CaMV 35S promoter. Plant Physiology, June 1995, Volume 108, No. 2, discloses in Abstract 803, the expression of heterologous genes following electroporation of the marine diatom. Electroporation induced loading of plasmid CaMV35S.
The engineering of novel traits into plants and other crops promises to be an area of great agricultural importance (Maiti and Hunt, 1992; Wagner, 1992). Plant genetic engineering techniques allow researchers to introduce heterologous genes of interest into plants cells to obtain the desired qualities in the plants of choice. Plant genetic engineering has led to a rapid progress in production of economically valuable germplasm with improved characteristics or traits such as insect resistance, virus resistance, fungal resistance, herbicide resistance, bacterial or nematode pathogen resistance, cold or drought tolerance, improved nutritional value, seed oil modification, delayed ripening of fruits, and male sterility, to name a few.
These newly created germplasms provide a enhanced development in breeding programs for crops improvement as well as a better understanding of gene regulation and organization in transgenic plants. The expression of useful foreign traits in plants is a major focus in plant biotechnology. Plant metabolic engineering is the application of genetic engineering methods to modify the nature of chemical metabolites in plants. For metabolic engineering where multiple genes need to be inserted into one cell, the use of different strong constitutive promoters is desirable in order to avoid genetic instability caused by recombination between identical or closely related promoter sequences taken from plants themselves. Through use of the present promoter sequence the introduced genes can be transcribed to messenger RNA and then translated to resultant proteins to exhibit new traits or characters.
Besides developing useful traits in crops, the present invention provides a further understanding of molecular pathways involved in disease development and secondary metabolism in plants. Moreover, by engineering plants with specific foreign genes, the responses of plants to abiotic and biotic stress and stress related metabolism can be analyzed. The invention described herein in developing gene vectors with newly defined promoters of the caulimoviruses advances this effort.
A wide variety of well-characterized genes of animal, human, bacterial and of plant origin, including those of several viruses, are available for engineering plants. For the most effective expression of this wide selection of genes either constitutive or regulated, versatile gene expression vectors are required. At the University of Kentucky, Dr. Arthur Hunt and his colleagues have developed a series of plant expression vectors (Schardl, at al., 1987) with a constitutive 35S promoter from cauliflower mosaic virus (CaMV) which have been successfully used to produce transgenic plants (Maiti, et al., 1988, 1989, 1991, 1993, 1994, 1995; Graybosh, et al., 1989; Berger, et al., 1989; Yeargan, et al., 1992; Liod, et al., 1992).
The present invention, develops additional useful promoters from FMV for high level expression of foreign genes in transgenic tobacco. These vectors are useful for both direct DNA uptake by isolated protoplasts and Ti plasmid- mediated gene transfer.
Enhanced levels of transcription via highly active promoters are essential for high levels of gene expression. The most widely used promoter for plant transformation, as described earlier, has been the 35S promoter of CaMV. It is active in a wide variety of plants and tissues. It is also the most thoroughly characterized promoter with respect to the sequence elements active in its transcriptional activity (Benfey and Chua, 1990). Kay, et al., 1987 showed that the transcriptional activity of the CaMV 35S promoter could be increased approximately tenfold by making a tandem duplication of 250 base pairs of upstream sequence.
Similar observation have been made with other promoters (McNeall, et al., 1989). The present inventors have constructed and tested a construct with the FMV FLt promoter.
The Monsanto Co. has recently patented 35S and the 19S promoters of CaMV, and the full length transcript promoter from FMV. In both cases cloned DNA material was provided to Monsanto Co. by the present investigator, Dr. Shepherd, University of Kentucky, Lexington, Ky. The present inventors have overcome the deficiencies of prior transgenic plant promoters and have now developed new, unique promoters of equal or better expression strength.